1. Field of the Invention
The present invention relates to a xcex2-fructofuranosidase gene, a process for isolating the gene, and a system for producing a xcex2-fructofuranosidase. More particularly, the present invention relates to a novel xcex2-fructofuranosidase, a DNA encoding it, and a process for isolating a DNA encoding xcex2-fructofuranosidase; a novel mold fungus having no xcex2-fructofuranosidase and a process for producing a recombinant xcex2-fructofuranosidase using the mold fungus as a host; and a xcex2-fructofuranosidase variant which selectively and efficiently produces a specific fructooligosaccharide such as 1-kestose from sucrose.
2. Description of the Related Art
The molecular structure of a fructooligosaccharide is the same as that of sucrose, except that the fructose half of a fructooligosaccharide is coupled with another one to three fructose molecules at positions C1 and C2 via a B bond. Fructooligosaccharides are indigestible sugars known for their physiological advantages, such as the facilitation of Bifidobacterial growth in the intestines, metabolic stimulation for cholesterols and other lipids, and little cariosity.
Fructooligosaccharides are found in plants, such as asparagus, onion, Jerusalem-artichoke and honey. They are also synthesized from sucrose by the newly industrialized mass production technique using fructosyltransfer reaction which is catalyzed by a xcex2-fructofuranosidase derived from a microorganism. However, as xcex2-fructofuranosidase preparations which are currently used for the industrial production of fructooligosaccharides is a cell-bound xcex2-fructofuranosidase derived from Asperglllus niger. They contain a relatively large proportion of proteins as impurities. Therefore, a need still exists for a high-purity xcex2-fructofuranosidase preparation with little unwanted proteins and a high titer. Further, an extracellular xcex2-fructofuranosidase is desired in an attempt to improve efficiently by using it in a fixed form, as an extracellularly available enzyme is more suitable for fixation.
Genes encoding xcex2-fructofuranosidase have been isolated from bacteria (Fouet, A., Gene, 45, 221-225 (1986), Martin, I. et al., Mol. Gen. Genet., 208, 177-184 (1987), Steininctz, M. et al., Mol. Gen. Genet., 191, 138-144 (1983), Scholle, R. et al., Gene, 80,49-56 (1989), Aslanidis, C. et al., J. Bacteriol., 171, 6753-6763 (1989), Sato, Y. and Kuramitsu, H. K., Infect. Immun., 56, 1956-1960 (1989), Gunasekaran, P. et al., J. Bacteriol., 172, 6727-6735 (1990)); yeast (Taussing, R, and M. Carlson, Nucleic Acids Res., 11, 1943-1954 (1983), Laloux, O. et al., FEBS Lett., 289, 64-68 (1991); mold (Boddy, L. M. et al., Curr, Genet., 24, 60-66 (1993); and plants (Arai, M. et al., Plant Cell Physiol., 33, 245-252 (1992), Unger, C. et al. Plant Physiol., 104, 1351-1357 (1994), Elliott, K. et al., Plant Mol. Biol., 21, 515-524 (1993), Sturm, A. and Chrispeels, M. J., Plant Cell, 2, 1107-1119 (1990)). However, to the best knowledge of the inventors, no gene has been found which encodes a xcex2-fructofuranosidase having transferase activity and is usable for the industrial production of fructooligosaccharides.
If a xcex2-fructofuranosidase gene usable for the industrial production of fructooligosaccharides is obtained, other functionally similar genes may be isolated, making use of their homology to the former. To the best knowledge of the inventors, no case has been reported on the screening of a new xcex2-fructofuranosidase gene using this technique. A process for isolating a xcex2-fructofuranosidase gene by this approach may also be applied to the screening of xcex2-fructofuranosidase enzyme to achieve significantly less effort and time than in conventional processes: first, using a xcex2-fructofuranosidase gene as a probe, a similar xcex2-fructofuranosidase gene is isolated, making use of its homology to the former; then, the isolated gene is introduced and expressed in a host which does not metabolize sucrose, such as Trichoderma viride, or a mutant yeast which lacks sucrose metabolizing capability (Oda, Y. and Ouchi, K., Appl. Environ. Microbiol., 1989, 55, 1742-1747); a homogeneous preparation of xcex2-fructofuranosidase is thus obtained as a genetic product with significantly less effort and time of screening. Furthermore, if the resultant xcex2-fructofuranosidase exhibits desirable characteristics, its encoding gene may be introduced in a safe and highly productive strain to enable the production of the desired xcex2-fructofuranosidase.
In addition, for producing such desirable xcex2-fructofuranosidase, designing a system for production, particularly a host which does not metabolize sucrose, is an important consideration. Using a host which intrinsically has xcex2-fructofuranosidase activity would result in a mixture of the endogenous xcex2-fructofuranosidase of the host and the xcex2-fructofuranosidase derived from the introduced gene. In this case, to take advantage of the xcex2-fructofuranosidase derived from the introduced gene, it must be isolated from the endogenous xcex2-fructofuranosidase of the host before application. On the contrary, using a host which lacks xcex2-fructofuranosidase activity would eliminate the need for enzyme isolation. In other words, the resultant unpurified enzyme would show the desirable characteristics of the xcex2-fructofuranosidase derived from the introduced gene. Known examples of microorganisms which do not have xcex2-fructofuranosidase activity include the Trichoderma strains and yeast mutants lacking sucrose metabolizing capability (Oda, Y. Ibid.) as described above. However, considering that the resultant xcex2-fructofuranosidase will be applied in food industry, a better candidate for a host would be a strain having no xcex2-fructofuranosidase selected from Aspergillus mold fungi which have been time-tested for safety through application to foods and industrial production of enzymes.
Furthermore, if a xcex2-fructofuranosidase gene usable for the industrial production of fructooligosaccharides is obtained, it may enable the development of a mutant with improved characteristics. For example, xcex2-fructofuranosidase which produces 1-kestose selectively and efficiently would provide the following advantage:
The molecular structures of 1-kestose and nystose, which make up part industrially produced fructooligosaccharide mixtures of today, are the same as that of sucrose except that their fructose half is coupled with one and two molecules of fructose, respectively. It has been found recently that their high-purity crystals exhibit new desirable characteristics both in physical properties and food processing purpose while maintaining the general physiological advantages of fructooligosaccharides (Japanese Patent Application No. 222923/1995, Japanese Patent Laid-Open Publication No. 31160/1994). In this sense, they are fructooligosaccharide preparations having new features.
In consideration of the above, some of the inventors have proposed an industrial process for producing crystal 1-kestose from sucrose (Japanese Patent Application No. 64682/1996, Japanese Patent Application No.77534/ 1996, and Japanese Patent Application No. 77539/1996). According to this process, a xcex2-fructofuranosidase harboring fructosyltransferase activity is first allowed to act on sucrose to produce 1-kestose; the resultant 1-kestose is fractionated to a purity of 80% or higher by chromatographic separation; then, using this fraction as a crystallizing sample, crystal 1-kestose is obtained at a purity of 95% or higher. The xcex2-fructofuranosidase harboring fructosyltransferase activity used in this process should be able to produce 1-kestose from sucrose at a high yield while minimizing the byproduct nystose, which inhibits the reactions in the above steps of chromatographic separation and crystallization. In the enzyme derived from Aspergillus niger, which is currently used for the industrial production of fructooligosaccharide mixtures, the 1-kestose yield from sucrose is approximately 44%, while 7% is turned to nystose (Japanese Patent Application No. 64682/1996). These figures suggest that the enzyme has room for improvement in view of the industrial production of crystal 1-kestose. As a next step, new enzymes having more favorable characteristics were successfully screened from Penicillium roqueforti and Scopulariopsis brevicaulis. These enzymes were able to turn 47% and 55% of sucrose into 1-kestose, respectively, and 7% and 4% to nystose (Japanese Patent Application No. 77534/1996, and Japanese Patent Application No. 77539/1996). Although these figures show that the new enzymes were superior to the enzyme derived from Aspergillus niger for higher 1-kestose yields and less nystose production from sucrose, the productivity and stability of the enzymes were yet to be improved. Thus, it is awaited to see a new enzyme that maintains the productivity and stability of the enzyme derived from Aspergillus niger, which is currently used for the industrial production of fructooligosaccharide mixtures, while achieving a sucrose-to-1-kestose yield comparable or superior to that of the enzymes derived from Penicillium roqueforti and Scopulariopsis brevicaulis. 
The inventors have now successfully isolated a novel xcex2-fructofuranosidase gene, and developed a process for isolating other xcex2-fructofuranosidase genes using the novel gene.
The inventors have also successfully produced a novel mold fungus having no xcex2-fructofuranosidase activity, and developed a system for producing a recombinant xcex2-fructofuranosidase using the mold fungus as a host.
Further, the inventors have found that the characteristics of xcex2-fructofuranosidase with fructosyltransferase activity change with its amino acid sequence, and have successfully produced a xcex2-fructofuranosidase variant which selectively and efficiently produces a specific fructooligosaccharide such as 1-kestose from sucrose.
The present invention is based on these findings.
Thus, the first aspect of the present invention provides a novel xcex2-fructofuranosidase gene and a xcex2-fructofuranosidase encoded by the gene.
The second aspect of the present invention provides a process for isolating a xcex2-fructofuranosidase gene using the novel xcex2-fructofuranosidase gene. The process according to the second aspect of the present invention also provides a novel xcex2-fructofuranosidase.
In addition, the third aspect of the present invention provides a novel mold fungus having no xcex2-fructofuranosidase activity and a system for producing a recombinant xcex2-fructofuranosidase using the mold fungus as a host.
Further, the fourth aspect of the present invention provides a xcex2-fructofuranosidase variant which selectively and efficiently produces a specific fructooligosaccharide such as 1-kestose from sucrose.
The xcex2-fructofuranosidase according to the first aspect of the present invention has the amino acid sequence of SEQ ID No. 1 as shown in the sequence listing.
In addition, the xcex2-fructofuranosidase gene according to the first aspect of the present invention encodes the amino acid sequence of SEQ ID No. 1 as shown in the sequence listing.
Further, the process for isolating a xcex2-fructofuranosidase gene according to the second aspect of the present invention is a process for isolating a xcex2-fructofuranosidase gene, making use of its homology to a nucleotide sequence comprising all or part of the nucleotide sequence of SEQ ID No. 2 as shown in the sequence listing.
In addition, a novel xcex2-fructofuranosidase which has been isolated in the process according to the second aspect of the present invention is a polypeptide comprising the amino acid sequence of SEQ ID No. 11 or 13 as shown in the sequence listing or a homologue thereof.
Furthermore, the mold fungus according to the third aspect of the present invention is a mold fungus having no xcex2-fructofuranosidase by deleting all or part of the xcex2-fructofuranosidase gene on the chromosome DNA of the original Aspergillus mold fungus.
The xcex2-fructofuranosidase variant according to the fourth aspect of the present invention is a mutant xcex2-fructofuranosidase with fructosyltransferase activity obtained by a mutation in the original xcex2-fructofuranosidase thereof, wherein the variant comprises an insertion, substitution or deletion of one or more amino acids in, or an addition to either or both of the terminals of, the amino acid sequence of the original xcex2-fructofuranosidase, and the composition of the fructooligosaccharide mixture produced from sucrose as a result of fructosyltransfer reaction by the xcex2-fructofuranosidase variant differs from the composition of the fructooligosaccharide mixture produced by the original xcex2-fructofuranosidase.